Expression Of Endoplasmic Reticulum Stress Proteins Research Articles

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway.

BACKGROUNDChronic biliary obstruction results in ischemia and hypoxia of hepatocytes, and leads to apoptosis. Apoptosis is very important in regulating the homeostasis of the hepatobiliary system. Endoplasmic reticulum (ER) stress is one of the signaling pathways that induce apoptosis. Moreover, the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-induced apoptotic pathway is the main way; but its role in liver injury remains unclear. Yinchenhao decoction (YCHD) is a traditional Chinese medicine formula that alleviates liver injury and apoptosis, yet its mechanism is unknown. We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice (OJ).AIMTo investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein (CHOP)-growth arrest and DNA damage-inducible protein 34 (GADD34) pathway and B cell lymphoma/leukemia-2 related X protein (Bax)/B cell lymphoma/leukemia-2 (Bcl-2) ratio.METHODSFor in vivo experiments, 30 rats were divided into three groups: control group, OJ model group, and YCHD-treated group. Blood was collected to detect the indicators of liver function, and liver tissues were used for histological analysis. For in vitro experiments, 30 rats were divided into three groups: G1, G2, and G3. The rats in group G1 had their bile duct exposed without ligation, the rats in group G2 underwent total bile duct ligation, and the rats in group G3 were given a gavage of YCHD. According to the serum pharmacology, serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells. Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling (TUNEL) assay was used to detect BRL-3A hepatocyte apoptosis. Alanine aminotransferase (ALT) and aspartate transaminase (AST) levels in the medium were detected. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were used to detect protein and gene expression levels of PERK, CHOP, GADD34, Bax, and Bcl-2 in the liver tissues and BRL-3A cells.RESULTSBiochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group. The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ. Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ. Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK, CHOP, and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ. The Bax and Bcl-2 levels were increased, and the Bax/Bcl-2 ratio was also increased. When YCHD was used, the PERK, CHOP, GADD34, and Bax levels quickly decreased, while the Bcl-2 levels increased, and the Bax/Bcl-2 ratio decreased.CONCLUSIONOJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio. YCHD can attenuate these changes.

ERdj5 in Innate Immune Cells Is a Crucial Factor for the Mucosal Adjuvanticity of Cholera Toxin.

Cholera toxin (CT) is one of most strong mucosal adjuvants, but it cannot be clinically used owing to its toxicity. The cytosolic A1 subunit of CT (CTA1) is the molecule responsible for its immunostimulatory activity, which increases the concentration of cyclic AMP and causes the induction of pro-inflammatory cytokines in innate immune cells. However, the importance of endoplasmic reticulum (ER) molecules involved in CTA1 retro-translocation to induce immune responses remained to be investigated. ERdj5 is an ER protein which is expected to transfer CTA1 to the Hrd1 complex for the retro-translocation of CTA1. In this study, we investigated the physiological relevance of ERdj5 in immune stimulation by CT. ERdj5-knockout (ERdj5 KO) mice had decreased production of antigen-specific IgG in the serum and IgA in the mucosal secretion after intranasal immunization with Ag and CT. Especially, IgG2c isotypes were specifically reduced in the absence of ERdj5. ERdj5 KO dendritic cells (DCs) failed to full activation with decreased expression of costimulatory molecules, such as MHC class II, CD80, and CD 86. In ERdj5 KO DCs, secretion of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, was reduced. The cytokine signatures of several helper T cells were reduced in ERdj5 KO mice following intranasal CT immunization. The absence of ERdj5 affects the immunostimulatory properties of CT but does not affect the response to the CTB pentamer, the response to alum, total antibody production, or cytokine release from DCs exposed to CpG. Interestingly, CT enhanced the expression of ER stress proteins in ERdj5 KO innate immune cells. These results suggested that ERdj5 contributed as a decisive factor to the immunostimulatory capacity of CT via CTA1 retro-translocation.

Parkinson\u2019s disease-associated LRRK2-G2019S mutant acts through regulation of SERCA activity to control ER stress in astrocytes

Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a common feature of Parkinson’s disease (PD) and further suggests that several PD-related genes are responsible for ER dysfunction. However, the underlying mechanisms are largely unknown. Here, we defined the mechanism by which LRRK2-G2019S (LRRK2-GS), a pathogenic mutation in the PD-associated gene LRRK2, accelerates ER stress and cell death. Treatment of cells with α-synuclein increased the expression of ER stress proteins and subsequent cell death in LRRK2-GS astrocytes. Intriguingly, we found that LRRK2-GS localizes to the ER membrane, where it interacts with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and suppress its activity by preventing displacement of phospholamban (PLN). LRRK2-GS–mediated SERCA malfunction leads to ER Ca2+ depletion, which induces the formation of mitochondria-ER contacts and subsequent Ca2+ overload in mitochondria, ultimately resulting in mitochondrial dysfunction. Collectively, our data suggest that, in astrocytes, LRRK2-GS impairs ER Ca2+ homeostasis, which determines cell survival, and as a result, could contribute to the development of PD.

Inhibition of Brd4 alleviates renal ischemia/reperfusion injury-induced apoptosis and endoplasmic reticulum stress by blocking FoxO4-mediated oxidative stress

Ischemia/reperfusion injury (I/R) is one of the leading causes of acute kidney injury (AKI) that typically occurs in renal surgeries. However, renal I/R still currently lacks effective therapeutic targets. In this study, we proved that inhibition of Brd4 with its selective inhibitor, JQ1, could exert a protective role in renal I/R injury in mice. Inhibiting Brd4 with either JQ1 or genetic knockdown resulted in reduction of endoplasmic reticulum stress (ERS)-associated protein and proapoptotic protein expression both in I/R-induced injury and hypoxia/reoxygenation (H/R) stimulation in HK-2 cells. H/R-induced apoptosis and ERS depended on oxidative stress in vitro. Moreover, FoxO4, which is involved in the generation of hydrogen peroxide, was up-regulated during H/R stimulation-mediated apoptosis and ERS, and this upregulation could be abolished by Brd4 inhibition. Consistently, FoxO4-mediated ROS generation was attenuated upon inhibition of Brd4 with JQ1 or siRNA against Brd4. Further, the transcriptional activity of FoxO4 was suppressed by PI3K and AKT phosphorylation, which are upstream signals of FoxO4 expression, and were enhanced by Brd4 both in vivo and in vitro. In conclusion, our results proved that Brd4 inhibition blocked renal apoptotic and ERS protein expression by preventing FoxO4-dependent ROS generation through the PI3K/AKT pathway, indicating that Brd4 could be a potential therapeutic target for renal I/R injury.

Interventional effect of bicyclol on isoniazid-induced liver injury in rats and the expression of glucose-regulated protein 78, and growth arrest and DNA-damage-inducible gene 153

Objective: To investigate the interventional effect of bicyclol on isoniazid-induced liver injury in rats and the expression of endoplasmic reticulum stress (ERS) protein, glucose regulatory protein 78 (GRP78), and growth arrest and DNA-damage-inducible gene 153(CHOP). Methods: Eighty Wistar rats were randomly divided into control group (8 rats) and model group (72 rats). After 10 days of intragastric administration of isoniazid, the model group rats were randomly divided into treatment group (A), natural recovery group (B), etiological persistence group (C) and etiological persistence plus treatment group (D). Sixteen rats from each group were sacrificed after 1 and 2 weeks of intervention with different methods. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected. Liver pathological morphology was observed. Apoptotic cells were detected by TUNEL assay. ERS protein expression was detected by Western blot. A t-test or randomized block analysis of variance, K-S test and Levene's test were used to analyze the normality and homogeneity of variance. Kruskal-Wallis rank sum test was used for data that did not suit the conditions of t-test and variance analysis. Results: ALT and AST were elevated in the model group, and liver pathological examination showed liver tissue damage. Apoptotic index was higher than control group (7.13% ± 1.55% vs. 0.75% ± 0.71%, Z = -3.411, P < 0.01), and the expression value of ERS protein in model group was significantly higher than control group (GRP78: 1.16 ± 0.30 vs. 0.23 ± 0.05, t = -6.008, P < 0.01; CHOP: 0.98±0.23 vs. 0.20 ± 0.10, t = -6.378, P < 0.01). Serum enzymes, apoptotic index and ERS protein expressions of rats were decreased after treatment with bicyclol, and the pathological damage was eased. Rats in natural recovery group recovered less than the treatment group. Conclusion: Isoniazid-induced liver injury is associated to ERS-related excessive apoptosis and the therapeutic effect of bicyclol on drug-induced liver injury may minimize ERS-induced apoptosis.

Glucagon-like peptide-1 attenuates endoplasmic reticulum stress-induced apoptosis in H9c2 cardiomyocytes during hypoxia/reoxygenation through the GLP-1R/PI3K/Akt pathways.

Endoplasmic reticulum (ER) stress-induced apoptosis is a major cause of myocardial ischemia/reperfusion (I/R) injury. Emerging evidence indicates that glucagon-like peptide-1 (GLP-1) has potential cardioprotective effects. However, the precise mechanisms underlying the involvement of GLP-1 in I/R injury remain largely unknown. In the present study, we aimed to determine whether GLP-1 attenuates hypoxia/reoxygenation (H/R) injury in cardiomyocytes and to further elucidate the underlying signaling pathway. The results indicate that GLP-1 reversed the increased apoptotic ratio, the increased lactate dehydrogenase (LDH) levels, the reduced cell viability, the increased Caspase-3 activity, and the increased Bax/Bcl-2 ratio caused by H/R. Importantly, GLP-1 significantly decreased the expression of H/R-induced ER stress proteins (GRP78, CHOP) and Caspase-12. In addition, we found that GLP-1 increased the expression of p-Akt in H9c2 cells with H/R injuries, and that the protective action of GLP-1 against H/R-induced injury was blocked by the GLP-1 receptor (GLP-1R) inhibitor Exendin9-39 and the PI3K inhibitor LY294002. Exendin9-39 and LY294002 also blocked the downregulation of ER stress protein expression by GLP-1, after H/R injury. Therefore, we have shown that GLP-1 exerts its cardioprotective effects by alleviating ER stress-induced apoptosis due to H/R injury and that these effects are most likely associated with the activation of GLP-1R/PI3K/Akt signaling pathway.

Effects of calpain inhibitor on the apoptosis of hepatic stellate cells induced by calcium ionophore A23187.

We previously showed that changes in calcium concentrations were related to cell apoptosis in vitro. The endoplasmic reticulum (ER) is the main component of calcium storage and signal transduction, and disrupting the balance of intracellular Ca2+ can cause endoplasmic reticulum stress (ERS). In this process, the ER releases stored Ca 2+ into the cytoplasm and activates calpain-2. To further investigate the effect of calpain in hepatic stellate cells (HSCs), in the current study, we examine the effect of N-acetyl-leu-leu-norleucinal (ALLN) on apoptosis resulting from calcium ionophore A23187-induced ERS. Our findings indicate that calpain inhibition reduces calcium ionophore A23187-induced apoptosis of HSCs and decreases the expression of ER stress proteins that may be related to the calpain/caspase signaling pathway.

Melatonin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress plays a vital role in mediating ischemic reperfusion damage in brain. In this study, we evaluated whether melatonin inhibits ER stress in cultured neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to transient focal cerebral ischemia. Sprague-Dawley rats were treated with melatonin (5 mg/kg) or control at reperfusion onset after transient occlusion of the right middle cerebral artery (MCA) for 90 min. Brain infarction and hemorrhage within infarcts were measured. The expression of ER stress proteins of phosphorylation of PRKR-like endoplasmic reticulum kinase (p-PERK), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by western blotting and immunohistochemistry analysis. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method, cleaved caspase-3 and cytochrome c were used to investigate cell apoptosis in OGD-induced cultured neurons. Our results demonstrated that animals treated with melatonin had significantly reduced infarction volumes and individual cortical lesion sizes as well as increased numbers of surviving neurons. Melatonin can significantly modulate protein levels by decreasing both p-PERK and p-eIF2α in the ischemic core and penumbra. Moreover, the expressions of ATF4 and CHOP were restrained in the ischemic core and penumbra, respectively. Furthermore, pretreatment with melatonin at 10–100 µM effectively reduced the levels of p-PERK and p-eIF2α in cultured neurons after OGD injury. Melatonin treatment also effectively decreased neuron apoptosis resulting from OGD-induced neuron injury. These results indicate that melatonin effectively attenuated post-ischemic ER stress after ischemic stroke.

Ischemic preconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PERK pathway.

The purpose of this study was to explore the effects of cerebral ischemic preconditioning which can decrease brain ischemia/reperfusion (I/R) injury by inhibiting endoplasmic reticulum (ER) stress-induced apoptosis. The focal cerebral ischemia rat was selected as the experimental model. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in ischemic penumbra were assessed after cerebral reperfusion. We assessed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells and measured the expressions of phosphorylation PERK (p-PERK), glucose-regulated protein 78 (GRP78), activating transcription factor-4 (ATF4) and caspase-12 in ischemic penumbra after cerebral reperfusion. We showed that the infarct sizes can be reduced due to the preconditioning under the influence of brain ischemia after reperfusion. The effect of preconditioning on the expression of ER stress proteins suggested the expressions of the 4 proteins p-PERK, ATF4, caspase-12 and GRP78 in the penumbra cortex by immunohistochemistry and Western blot increased after cerebral ischemia. Significant reduction of the number of TUNEL-positive cells was in the penumbra cortex of the preconditioning group. We found that cerebral ischemic preconditioning can protect the brain from I/R injury by inhibiting ER stress-induced apoptosis; the pathway of PERK is involved.

Calcium homeostasis and endoplasmic reticulum stress are involved in Salvianolic acid B-offered protection against cardiac toxicity of arsenic trioxide.

Arsenic trioxide (ATO) is a potent anticancer agent used to treat acute promyelocytic leukemia. However, its cardiotoxicity limits ATO’s widespread clinical use. Previous studies demonstrated that ATO may aggravate Ca2+ overload and promote endoplasmic reticulum stress (ERS). Salvianolic acid B (Sal B) is cardioprotective against ATO and enhances ATO’s anticancer activities. The present study assessed whether the Sal B protective effect was related to maintenance of Ca2+ homeostasis and inhibition of ER stress. Male BALB/c mice were injected with ATO or ATO+Sal B once a day via the tail vein for 2 weeks. We then detected the effects of Sal B in real time using adult rat ventricular cardiomyocytes in vitro using an IonOptix MyoCam system. Sal B treatment alleviated ATO-induced abnormal cardiac contractions and Ca2+ homeostasis imbalance. Sal B increased sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity, regulated Ca2+ handling protein expression, and decreased expression of ERS proteins. Our results demonstrate that the cardioprotective effect of Sal B correlates with SERCA modulation, maintenance of Ca2+ homeostasis, and inhibition of ER stress. These findings suggest Sal B may ameliorate ATO cardiotoxicity during clinical application.

Gaseous signalling molecule SO2 via Hippo‑MST pathway to improve myocardial fibrosis of diabetic rats.

Recent studies have indicated the existence of an endogenous sulfur dioxide (SO2)-generating system in the cardiovascular system. The present study aimed to discuss the function and regulatory mechanism of gaseous signal molecule SO2 in inhibiting apoptosis and endoplasmic reticulum stress (ERS) via the Hippo-MST signaling pathway to improve myocardial fibrosis of diabetic rats. A total of 40 male Sprague-Dawley rats were randomly divided into four groups (10 rats per group): Normal control group (control group), diabetic rats group [streptozotocin (STZ) group], SO2 intervention group (STZ+SO2 group) and diabetes mellitus rats treated with L-Aspartic acid β-hydroxamate (HDX) group (HDX group). Diabetic rats models were established by intra-peritoneal injection of STZ (40 mg/kg) Following model establishment, intra-peritoneal injection of Na2SO3/NaHSO3 solution (0.54 mmol/kg) was administered in the STZ+SO2 group, and HDX solution (25 mg/kg/week) was administered in the HDX group. A total of 4 weeks later, echocardiography was performed to evaluate rats' cardiac function; Masson staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and transmission electron microscopy examinations were performed to observe myocardial morphological changes. ELISA was employed to determine the SO2 content. Western blot analysis was performed to detect the expression of proteins associated with apoptosis, ERS and the Hippo-MST signalling pathway. Compared with the control group, the STZ group and HDX group had a disordered arrangement of myocardial cells with apparent myocardial fibrosis, and echocardiography indicated that the cardiac function was lowered, there was an obvious increase of apoptosis in myocardial tissue, the expression levels of apoptosis-associated protein B-cell lymphoma associated protein X, caspase-3 and caspase-9 were upregulated, and Bcl-2 expression was downregulated. The expression of ERS and Hippo-MST pathway-associated proteins, including CHOP, GRP94, MST1 and MST2, were significantly upregulated. By contrast, these above-mentioned changes were reversed by SO2 treatment. Compared with STZ group, the HDX group had a further increase of myocardial fibrosis and apoptosis, while there were no statistically significant differences in the expression of Bax/Bcl-2, caspase-3, caspase-9 and ERS and Hippo-MST pathway-associated proteins. The results of the present study demonstrated that the gaseous signal molecule SO2 can effectively improve the myocardial fibrosis of diabetic rats, and its mechanism may be associated with reduced apoptosis and ERS by downregulated Hippo-MST pathway.

The role of the endoplasmic reticulum stress response following cerebral ischemia.

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods Invivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. Invitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results Invivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. Invitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection invitro, highlighting their complex role in neuronal biology following ischemia.

Enhanced Susceptibility to 5-Fluorouracil in Human Colon Cancer Cells by Silencing of GRP78.

Glucose-regulated protein (GRP78), also known as immunoglobulin heavy chain binding protein and as heat shock 70 kDa protein 5, is present in the endoplasmic reticulum (ER) membrane. GRP78 is generally expressed at low concentrations, but is increased by physiological stress. GRP78 is thought to protect against tissue or organ damage under pathological conditions, such as neurotoxic stress, myocardial infarction, or arteriosclerosis. In addition, in tumors, GRP78 expression is much higher than in normal tissues. Furthermore, high levels of GRP78 expression have been shown to increase the risk of malignancy and metastasis in prostate and colon cancer. Because both anticancer drugs and down-regulation of GRP78 expression inhibit cancer progression and growth, we hypothesized that down-regulation of GRP78 expression might lead to enhanced susceptibility of cancer cells to cytotoxic action of 5-fluorouracil (5-FU). GRP78 expression was suppressed in LoVo colon cancer cells by utilizing small-interfering RNA (si-GRP78), and the cells were subsequently used to study the antiproliferative and anticancer effects of 5-FU treatment. The signaling pathways responsible for the increase of LoVo cell susceptibility to 5-FU treatment after exposure to GRP78 siRNA were determined by western blot. GRP78 silencing significantly inhibited cell viability and increased apoptosis of LoVo cells. Furthermore, combined treatment with 5-FU and GRP78 siRNA for 12 h reduced cell viability, and increased apoptosis and generation reactive oxygen species more strongly than either of the two treatments applied separately. In order to examine the role of ER stress in increased susceptibility of LoVo cells to 5-FU after pretreatment with GRP78 siRNA, we analyzed expression levels of ER stress marker proteins, such as phosphorylated protein kinase-like endoplasmic reticulum kinase (PERK), phosphorylated eukaryotic initiation factor 2 alpha (eIF2α), activating transcription factor 4 (ATF4), phosphorylated inositol-requiring enzyme 1 alpha (IRE1α), phosphorylated p38, and C/EBP homologous protein (CHOP). Treatment with 5-FU alone increased the expression of ER stress marker proteins, whereas combined exposure to both 5-FU and GRP78 siRNA led to an even stronger effect on these markers. Similar to the pattern of modulation of ER stress protein expression, the levels of apoptosis-related proteins were also more strongly affected by combined exposure to 5-FU and GRP78 siRNA than by single treatments. In particular, expression of Bcl-2-associated X protein (BAX), cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase 1 (PARP1) were increased, whereas the expression of B-cell lymphoma 2 (BCL2) was reduced by these treatments. GRP78 silencing and incubation with 5-FU have synergistic effects on the inhibition of LoVo colon cancer cell growth via the induction of ER stress-dependent apoptosis.

Effects of electroacupuncture combined with dietary control on liver endoplasmic reticulum stress in rats with non-alcoholic fatty liver disease

To observe the effects of electroacupuncture (EA) combined with dietary control on liver endoplasmic reticulum stress (ERS) in rats with non-alcoholic fatty liver disease (NAFLD), and to explore the action mechanism of EA on NAFLD. Sixty male SD rats were randomly divided into a common diet group (15 rats) and a model group (45 rats, fed with high-fat diet to induce NAFLD model). After model establishment, 10 rats in the common diet group were randomly selected as a control group, and 40 rats in model group were randomly divided into a high-fat diet (HFD) group, a low-fat diet (LFD) group, an EA-1 group and an EA-2 group, 10 rats in each one. From the 2nd day of model establishment, rats in the HFD group and EA-1 group were fed with high-fat diet, while rats in the LFD group and EA-2 group were fed with common diet. Rats in the EA-1 group and EA-2 group were treated with acupuncture at unilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Taichong" (LR 3), besides,"Zusanli" (ST 36) and "Sanyinjiao" (SP 6) were connected with EA. The treatment was given once a day, and two sides of acupoints were selected alternatively. Each treatment last 20 min for totally 4 weeks. The rats in the remaining groups were treated with identical fixation method. After treatment, the liver tissues of rats in each group were collected and made into ultra-thin slice to perform transmission electron microscope (TEM) on the endoplasmic reticulum morphology; western blot and real-time PCR method were applied to observe the protein and gene expression of the specific ERS protein, glucose-regulated protein 78 (GRP78). Under TEM, plenty of rough endoplasmic reticulum in alignment was observed in the cytoplasm of rats in the control group, accompanied with indistinct adhering ribosome; the rough endoplasmic reticulum in the cytoplasm in the HFD group was in expansion and obvious breakage, presenting small vacuoles in irregular arrangement; the expansion of rough endoplasmic reticulum in the LFD group and EA-1 group was relieved without appa-rent breakage and disarrangement; the rough endoplasmic reticulum in the EA-2 group was in alignment without obvious expansion. The protein expression of GRP78 was little in the control group, while the protein and mRNA expression of GRP78 were obviously increased in the remaining groups (all P<0.01). The protein and mRNA expression of GRP78 in the LFD group and EA-1 group were significantly lower than those in the HFD group (all P<0.01); those in the EA-2 group were lower than those in the LFD group and EA-1 group (all P<0.01). EA effectively improves the liver ERS in NAFLD rats, which can be one of the action mechanisms of EA on NAFLD. In addition, the efficacy is more significant with diet control.

ABCG1 maintains high-grade glioma survival in vitro and in vivo.

The overall survival for adults with malignant glioma (glioblastoma) remains poor despite advances in radiation and chemotherapy. One of the mechanisms by which cancer cells develop relative resistance to treatment is through de-regulation of endoplasmic reticulum (ER) homeostasis. We have recently shown that ABCG1, an ATP-binding cassette transporter, maintains ER homeostasis and suppresses ER stress-induced apoptosis in low-grade glioma. Herein, we demonstrate that ABCG1 expression is increased in human adult glioblastoma, where it correlates with poor survival in individuals with the mesenchymal subtype. Leveraging a mouse model of mesenchymal glioblastoma (NPcis), shRNA-mediated Abcg1 knockdown (KD) increased CHOP ER stress protein expression and resulted in greater NPcis glioma cell death in vitro. Moreover, Abcg1 KD reduced NPcis glioma growth and increased mouse survival in vivo. Collectively, these results demonstrate that ABCG1 is critical for malignant glioma cell survival, and might serve as a future therapeutic target for these deadly brain cancers.

Spermine inhibits Endoplasmic Reticulum Stress - induced Apoptosis: a New Strategy to Prevent Cardiomyocyte Apoptosis

Background/Aims: Endoplasmic reticulum stress (ERS) plays an important role in the progression of acute myocardial infarction (AMI), in part by mediating apoptosis. Polyamines, including putrescine, spermidine, and spermine, are polycations with anti-oxidative, anti-aging, and cell growth-promoting activities. This study aimed to determine the mechanisms by which spermine protects against ERS-induced apoptosis in rats following AMI. Methods and Results: AMI was established by ligation of the left anterior descending coronary artery (LAD) in rats, and exogenous spermine was administered by intraperitoneal injection (2.5 mg/ml daily for 7 days pre-AMI). Spermine treatment limited infarct size, attenuated cardiac troponin I and creatinine kinase-MB release, improved cardiac function, and decreased ERS and apoptosis related protein expression. Isolated cardiomyocytes subjected to hypoxia showed significant increase in reactive oxygen species (ROS) and the expression of apoptosis and ERS related proteins; these effects occurred through PERK and eIF2α phosphorylation. The addition of spermine attenuated cardiomyocyte apoptosis, suppressed the production of ROS, and inhibited ERS related pathways. Conclusions: Spermine was an effective pre-treatment strategy to attenuate cardiac ERS injury in rats, and the cardioprotective mechanism occurring through inhibition of ROS production and down regulation of the PERK-eIF2α pathway. These findings provide a novel target for the prevention of apoptosis in the setting of AMI.

δV1-1 Reduces Pulmonary Ischemia Reperfusion-Induced Lung Injury by Inhibiting Necrosis and Mitochondrial Localization of PKCδ and p53

Ischemia-reperfusion (IR)-induced lung injury is one of the major contributing factors of morbidity and mortality after lung transplantation. To determine the IR-induced molecular changes in lung epithelial cells, we developed a cell-culture model that simulates lung preservation and transplantation. Six hours of cold ischemic time (CIT) and reperfusion elicited production of multiple inflammatory cytokines and chemokines and increased expression of endoplasmic reticulum (ER) proteins. Prolonged hypothermic condition (18 h CIT) reduced ER stress protein levels, and induced apoptosis and necrosis (via mechanisms related to mitochondrial permeability transition pore opening). Protein kinase C (PKCδ) was activated during CIT, and its downregulation via small interference (si) (in siRNA) RNA reduced IR-induced cytokine production and apoptotic cell death. δV1-1, a PKCδ peptide inhibitor, reduced translocation of PKCδ and p53 to the mitochondria after 18 h CIT, rescued ER stress protein expression, and converted the major mode of cell death from necrosis to apoptosis. Administration of δV1-1 effectively reduced lung transplantation and IR-induced pulmonary injury in rats. Therefore, inhibition of PKCδ by δV1-1 could be an effective strategy to ameliorate IR-induced lung injury by inhibiting the signaling pathways leading to necrosis.

C/EBP Homologous Protein-Mediated Endoplasmic Reticulum Stress-Related Renal Apoptosis Is Involved in Rats With Brain Death

BackgroundC/EBP homologous protein (CHOP) is an important marker in endoplasmic reticulum stress (ERS)-associated cell apoptosis. The role of CHOP-induced renal apoptosis remains unclear in rats with brain death (BD). The present study aims to investigate the possible implication of CHOP-mediated, ERS-related BD-induced apoptosis in rats. Materials and MethodsForty male Sprague–Dawley rats were divided randomly into 4 experimental groups. We examined activation of ERS and apoptosis-related protein expression using Western blot and immunohistochemical staining. In addition, apoptosis is assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. ResultsKidneys harvested after BD show increased ERS- and apoptosis-related protein markers compared with kidneys of non-BD rats. Salubrinal (Sal) significantly increased levels of p-eIF2a and decreased the activity of CHOP at 6 hours after BD compared with vehicle-treated dimethylsulfoxide. ConclusionsTreating the BD donor with Sal influences renal apoptosis compared with vehicle-treated BD rats. Our results indicate that targeting the CHOP pathway provides a promising therapeutic approach for kidney injury associated with donor BD.

Effect of sulfur dioxide on endoplasmic reticulum stress protein expression in myocardial tissue of rats with left to right shunt

Objective To explore the possible role of sulfur dioxide(SO2) in endoplasmic reticulum stress(ERS) protein expression in myocardial tissue of rats with left to right shunt. Methods A total of 24 male Sprague Dawley rats were randomly divided into sham group(n=8), shunt group(n= 8)and shunt plus SO2 donor group(n= 8). The rats of shunt group and shunt plus SO2 donor group, an abdominal aorta and inferior vena cava shunting was created.Na2SO3/NaHSO3 was administrated intra-peritoneally in rats of shunt plus SO2 donor group.Eight weeks later, the ratio of quantity of pulmonary to quantity of system(Qp/Qs) was measured; Heart weight/body weight(HW/BW) and right ventricle/left ventricle+ ventricular septum [RV/(LV+ SP)] were detected; Expressions of collagen type Ⅰ in right ventricle myocardial tissue was measured by enzyme linked immunosorbent assay(ELISA); High performance liquid chromatography was used to measure SO2 content in right ventricle myocardial tissue.Glucose regulated protein 78(GRP78), c-jun N-terminal kinase(JNK) and phosphorylation-c-jun N-termina kinase(p-JNK) mRNA and protein expressions in RV myocardial tissue were detected by Western blot and real time-polymerase chain reaction(RT-PCR). Results After 8-weeks shunting, Qp/Qs in the shunt and shunt plus SO2 groups were higher significantly than those of the sham group(all P<0.05). Compared with sham group, HW/BW, RV/LV+ SP and expressions of collagen type Ⅰ in the shunt group were significantly higher(all P<0.05). Treatment of SO2, HW/BW and RV/LV+ SP and collagen type Ⅰ in the shunt group were lower significantly(all P<0.05). The level of SO2 of RV in shunt group increased significantly compared with sham group(all P<0.05), but significantly lower than that of shunt plus SO2 donor group(P<0.05). Compared with sham group, the expressions of GRP78, JNK and p-JNK mRNA and protein in RV increased significantly in shunt group(P<0.05). After treatment with SO2, the expressions of GRP78, JNK and p-JNK mRNA and protein decreased significantly(P<0.05). Conclusion SO2 might protect RV myocardial tissue of the rats with left-right shunt by regulating the expressions of ERS. Key words: Sulfur dioxide; Endoplasmic reticulum stress; Glucose regulated protein 78; c-jun N-terminal kinase; Phosphorylation-c-jun N-terminal kinase

Expression of Endoplasmic Reticulum Stress Markers GRP78 and CHOP Induced by Oxidative Stress in Blue Light-Mediated Damage of A2E-Containing Retinal Pigment Epithelium Cells

Aims: Age-related lipofuscin N-retinylidene-N-retinylethanolamine (A2E) accumulated in human retinal pigment epithelium (RPE) cells confers susceptibility to blue light-mediated damage, which represents one pathogenesis of age-related macular degeneration. This study investigated the expression of 2 best-characterized endoplasmic reticulum (ER) stress markers, glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP), as well as their regulation by oxidative stress after blue light-mediated damage of A2E-containing RPE cells. Methods: ARPE-19 cells were incubated with A2E (10, 25, 50 μ<smlcap>M</smlcap>) for 2 h and exposed to blue light for 20 min. A2E distributions in RPE cells were assessed via laser scanning confocal microscope and liquid chromatography-mass spectrometry. Cell viability was measured by a Cell Titer 96 Aqueous One Solution cell proliferation assay. The quantity of intracellular reactive oxygen species (ROS) was detected by dihydroethidium fluorescence using flow cytometry. Expressions of GRP78 and CHOP were measured at both mRNA and protein levels. To examine the role of oxidative stress in regulating GRP78 and CHOP expression, RPE cells were pretreated with the antioxidant N-acetylcysteine (NAC) for 2 h. RNA interference of GRP78 performed by short hairpin RNA was used to evaluate the effect of GRP78 in blue light-mediated damage of RPE cells. Results: After blue light exposure, A2E-treated RPE cells showed a gradual decrease in cell viability and a particular increase in ROS levels. Meanwhile, the expressions of GRP78 and CHOP in A2E-treated RPE cells were significantly increased at different time points after illumination. Pretreatment with NAC attenuated the expression of 2 ER stress markers, especially CHOP in A2E and blue light-treated RPE cells. Silencing of GRP78 by RNA interference upregulated CHOP and caspase-12 expression as well as aggravated the blue light-mediated damage of A2E-laden RPE cells. Conclusion: RPE cells exhibited ROS accumulation and subsequent elevation of GRP78 and CHOP expression after A2E and blue light-induced damage. The ROS scavenger NAC diminished ER stress protein expression, suggesting a connection between ER and oxidative stress in blue light-mediated damage of A2E-containing RPE cells. Besides, GRP78 may play a protective role in it.